Epoxide polymer surfaces

ABSTRACT

Method and reagent composition for covalent attachment of target molecules, such as nucleic acids, onto the surface of a substrate. The reagent composition includes epoxide groups capable of covalently binding to the target molecule. Optionally, the composition can contain photoreactive groups for use in attaching the reagent composition to the surface. The reagent composition can be used to provide activated slides for use in preparing microarrays of nucleic acids.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.09/227,913, filed Jan. 8, 1999, which is a continuation-in-part of U.S.Ser. No. 08/940,213 filed Sep. 30, 1997, now U.S. Pat. No. 5,858,653,the entire disclosures of which are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates to reagents and support surfacesfor immobilization of biomolecules, such as nucleic acids and proteins.

BACKGROUND OF THE INVENTION

[0003] The immobilization of deoxyribonucleic acid (DNA) onto supportsurfaces has become an important aspect in the development of DNA-basedassay systems, including the development of microfabricated arrays forDNA analysis. See, for instance, “The Development of MicrofabricatedArrays of DNA Sequencing and Analysis”, O'Donnell-Maloney et al.,TIBTECH 14:401-407 (1996). Generally, such procedures are carried out onthe surface of microwell plates, tubes, beads, microscope slides,silicon wafers or membranes.

[0004] A commonly used method for immobilizing cDNA's or PCR productsinto arrays is to first coat glass slides with polylysine, then applythe DNA and illuminate with UV light to photocrosslink the DNA onto thepolylysine (for example, see Schena M, Shalon D, Heller R, Chai A, BrownP O, Davis R W, “Parallel Human Genome Analysis: Microarray-basedExpression Monitoring of 1000 Genes”, Proc. Natl. Acad. Sci. USA93(20):10614-9 (1996)). One disadvantage of this approach is that the UVcrosslinking causes undesirable damage to the DNA that is not all usefulfor the immobilization. Another disadvantages of this approach is thatUV crosslinking tends to be limited to longer nucleic acids (e.g., overabout 100-mers), as provided by cDNA's and PCR products (and in contrastto the shorter nucleic acids typically formed by synthesis and referredto as “oligonucleotides”). It appears that the potential damage inducedby UV radiation (e.g., the formation of thymine dimers) is simply toogreat, and/or the extent of immobilization is insufficient, to permitshorter nucleic acids to be used. A population of longer nucleic acids,however, even when crosslinked by UV, will typically provide ampleundamaged regions sufficient to permit accurate hybridization.

[0005] Only relatively few approaches to immobilizing DNA, to date, havefound their way into commercial products. One such product forimmobilizing oligonucleotides onto microwell plates is known as“NucleoLink™”, and is available from Nalge Nunc International (see,e.g., Nunc Tech Note Vol. 3, No. 17). In this product, the DNA isreacted with a carbodiimide to activate 5′-phosphate groups, which thenreact with functional groups on the surface. Disadvantages of thisapproach are that it requires the extra step of adding the carbodiimidereagent as well as a five hour reaction time for immobilization of DNA,and it is limited to a single type of substrate material.

[0006] As another example, Pierce has introduced a proprietary DNAimmobilization product known as “Reacti-Bind™ DNA Coating Solutions”(see “Instructions—Reacti-Bind™ DNA Coating Solution” January, 1997).This product is a solution that is mixed with DNA and applied tosurfaces such as polystyrene or polypropylene. After overnightincubation, the solution is removed, the surface washed with buffer anddried, after which it is ready for hybridization. Although the productliterature describes it as being useful for all common plastic surfacesused in the laboratory, it does have some limitations. For example,Applicants were not able to demonstrate useful immobilization of DNAonto polypropylene using the manufacturer's instructions. Furthermore,this product requires large amounts of DNA. The instructions indicatethat the DNA should be used at a concentration between 0.5 and 5 μg/ml.

[0007] Corning sells a product called “DNA-BIND™” for use in attachingDNA to the surface of a well in a microwell plate (see, e.g., theDNA-BIND™ “Application Guide”). The surface of the DNA-BIND™ plate iscoated with an uncharged, nonpolymeric, low molecular weight,heterobifunctional reagent containing an N-oxysuccinimide (NOS) reactivegroup. This group reacts with nucleophiles such as primary amines. Theheterobifunctional coating reagent also contains a photochemical groupand spacer arm which covalently links the reactive group to the surfaceof the polystyrene plate. Thereafter, amine-modified DNA can becovalently coupled to the NOS surface. The DNA is modified by adding aprimary amine either during the synthesis process to the nascentoligomer or enzymatically to the preformed sequence. Since the DNA-BIND™product is polystyrene based, it is of limited use for thoseapplications that require elevated temperatures such as thermal cycling.Corning also sells an aminosilane-coated glass slide, under thetradename CMT-GAPS™ coated slides, which uses the same protocol aspolylysine-coated slides for immobilizing DNA into microarrays.

[0008] TeleChem International, Inc. sells slides coated with an aldehydesilane as well as an aminosilane-coated slide. The aldehyde silaneslides have very high backgrounds when fluorescence is used fordetection. They also require an additional reduction step for stableimmobilization.

[0009] Finally, SurModics, Inc., the assignee of the present invention,has recently introduced a coated glass slide, under the tradename3D-Link™ that consists of a hydrophilic polymer containingamine-reactive ester groups immobilized onto the surface. For bestresults, this product also requires amine modification of the DNA to beimmobilized. As expected, however, the reactive ester groups tend to behydrolytically unstable, which limits the amount of time arrays can beprinted without some loss of performance to approximately eight hours.

[0010] The role of epoxide groups, in the course of binding orimmobilizing nucleic acids, has been described in various ways as well.For instance, Shi et al., U.S. Pat. No. 5,919,626 describe theattachment of unmodified nucleic acids to silanized solid phasesurfaces. The method involves the use of conventional nonpolymericreagents such as mercapto-silanes and epoxy-silanes which bond to thesurface by forming siloxane bonds with OH groups on the glass surface.

[0011] See also, U.S. Pat. No. 5,925,552 (Keogh, et al., “Method forAttachment of Biomolecules to Medical Devices Surfaces”), which providesa method for forming a coating of an immobilized biomolecule on asurface of a medical device to impart improved biocompatibility forcontacting tissue and bodily fluids. One such method includes convertinga biomolecule comprising an unsubstituted amide moiety into anamine-functional material, combining the amine-functional material witha medical device biomaterial surface comprising a chemical moiety (suchas, for example, an aldehyde moiety, an epoxide moiety, an isocyanatemoiety, a phosphate moiety, a sulphate moiety or a carboxylate moiety)which is capable of forming a chemical bond with the amine-functionalmaterial, to bond the two materials together to form an immobilizedbiomolecule on a medical device biomaterial surface. Included within thelong list of biomolecules described as being useful in this patent were“a DNA segment, a RNA segment, a nucleic acid” and others.

[0012] Also on the subject of epoxides, Nagasawa et al., (J. Appl.Biochem. 7:430-437, 1985) describe the use of Sepharoses activated withepichlorohydrin or bisoxirane (both of which provide epoxide groups) forimmobilizing DNA as immunosorbents for DNA antibodies. See also,Wheatley, et al., J. Chromatog. A 726:77-90 (1996) and Potuzak, et al.,Nucl. Acids Res. 5:297-303 (1978).

[0013] To date, however, there appears to be no description in the art,let alone commercial products, that provide an optimal combination ofsuch properties as hydrolytic stability, ease of use, minimized DNAdamage (due to exposure to crosslinking radiation), and the ability toimmobilize underivatized nucleic acids and/or shorter nucleic acidsegments. In turn, there appear to be no products presently available,nor descriptions in the art, that provide or suggest the ability to usepolymer-pendent epoxide groups adapted to immobilize either short orlong nucleic acids, let alone in both derivatized and underivatizedforms, and suitable for immobilization onto surfaces.

[0014] Finally, Surmodics, Inc., the assignee of the present invention,has previously described a variety of applications for the use ofphotochemistry, and in particular, photoreactive groups, e.g., forattaching polymers and other molecules to support surfaces. See, forinstance, U.S. Pat. Nos. 4,722,906, 4,979,959, 5,217,492, 5,512,329,5,563,056, 5,637,460, 5,714,360, 5,741,551, 5,744,515, 5,783,502,5,858,653, and 5,942,555.

SUMMARY OF THE INVENTION

[0015] The present invention provides a method and epoxide-based reagentcomposition for covalent attachment of target molecules onto the surfaceof a substrate, such as microscope slides, microwell plates, tubes,silicon wafers, beads or membranes. In a preferred embodiment, themethod and composition are used to immobilize nucleic acid probes ontomicroscope slides, e.g., for use in printing DNA microarrays. The methodand reagent of the present invention can be used to covalentlyimmobilize either derivatized (e.g., amine-derivatized) or underivatized(i.e., not having a group added for the purpose of thermochemicalreaction with an epoxide group) nucleic acids, and are particularlyuseful for underivatized nucleic acids. The immobilization method ofthis invention is very convenient to perform. In a preferred embodiment,the method involves the steps of coating a support with the reagent ofthis invention, printing the nucleic acid array, incubating the slide ina humid environment, blocking excess epoxide groups and washing theslide, after which it is ready for a hybridization assay.

[0016] Parent application U.S. Ser. No. 09/227,913, describes, interalia, a comprehensive method and reagent composition for covalentattachment of target molecules onto the surface of a substrate, using areagent that contains one or more thermochemically reactive groups(i.e., groups having a reaction rate dependent on temperature). Suitablegroups are selected from the group consisting of activated esters suchas N-oxysuccinimide (“NOS”), epoxide, azlactone, activated hydroxyl andmaleimide groups.

[0017] The present application is particularly concerned with reagentshaving epoxide groups, in the manner described above, and providesfurther examples and advantages concerning the use of such epoxide-basedreagents. Such advantages include, for instance, the ability to use thereagents to attach underivatized DNA, in addition to DNA derivatized tocontain amine or other reactive groups, as described in parentapplication U.S. Ser. No. 09/227,913. Such advantages also includeimproved resistance to hydrolysis demonstrated by epoxides, as comparedfor instance, to NOS groups.

[0018] The present invention provides a method for immobilizingbiomolecules, such as biopolymers selected from nucleic acids, proteins,and polysaccharides, the method comprising the steps of:

[0019] a) providing a solid support having a surface,

[0020] b) providing a reagent comprising one or more epoxide groups, andoptionally also comprising one or more photogroups,

[0021] c) coating the reagent on the support surface (e.g., covalentlyattaching the polymeric reagent to the support surface by activation ofthe photogroups),

[0022] d) providing a biopolymer having a corresponding thermochemicalreactive group,

[0023] e) attaching the biopolymer to the support by reacting itscorresponding reactive group with the bound epoxide group,

[0024] f) optionally blocking the remaining epoxide groups (e.g., usingan amine reagent), and

[0025] g) using the resultant coated support surface for its intendedpurpose, e.g., for the immobilization of biomolecules such as nucleicacids.

[0026] Applicants have found that polymers (and particularly hydrophilicpolymers) containing epoxide groups, of the type described herein, haveseveral advantages for DNA immobilization over previously used methods.These polymers, when coated onto silane-modified glass slides, forinstance, provide an improved method for immobilizing underivatized DNA.Therefore, using these reagents, it is not necessary to modify the DNAwith amines or other functional groups. Furthermore, the epoxide groupsare significantly more stable to hydrolysis than are the amine-reactiveester groups. Compared with UV crosslinking of DNA onto polylysine oraminosilane, the coated surfaces of this invention are more convenientto use and tend to result in fewer undesirable side reactions, therebyresulting in less modification of the DNA.

[0027] A reagent composition of the invention preferably provides one ormore epoxide (also known as “oxirane”) groups pendent on a polymericbackbone, such as a hydrophilic polyacrylamide backbone. Optionally, andpreferably, the reagent composition can also provide one or more pendentphotoreactive groups. The photoreactive groups (alternatively referredto herein as “photogroups”) can be used, for instance, to attach reagentmolecules to the surface of the support upon the application of asuitable energy source such as light. The epoxide groups, in turn, canbe used to form covalent bonds with appropriate functional groups on thetarget molecule.

[0028] Optionally, the composition and method of this invention can beprovided in the manner described in parent application U.S. Ser. No.08/940,213. In such an embodiment, the reagent composition can be usedfor attaching a target molecule to the surface of a substrate, andcomprises one or more groups for attracting the target molecule to thereagent, and one or more epoxide groups for forming covalent bonds withcorresponding functional groups on the attracted target molecule.Optionally, such a composition further provides photogroups for use inattaching the composition to a surface. In one embodiment, for instance,a plurality of photogroups and a plurality of ionic groups (e.g.,cationic groups) are attached to a hydrophilic polymer backbone. Thispolymer can then be coimmobilized with a second polymer backbone thatprovides the above-described epoxide groups for immobilization of targetmolecules. Suitable ionic groups include quaternary ammonium salts,protonated tertiary amines and other cationic groups such as phosphoniumcompounds. Also included are tertiary amine groups capable of beingprotonated when placed in an acid environment. Quaternary ammonium saltsinclude alkyl quaternary ammonium compounds, such as[3-(methacryloylamino)propyl]trimethylammonium chloride (MAPTAC), aswell as aromatic quaternary ammonium groups such as pyridiniumcompounds. Phosphonium compounds include polymers prepared from monomerssuch as tributyl(4-vinylbenzyl)phosphonium chloride, and are describedin J. Appl. Polymer Sci. 53:1237 (1994), the disclosure of which is alsoincorporated by reference.

[0029] The invention further provides a method of attaching a targetmolecule, such as a DNA molecule, to a surface, by employing a reagentas described herein. In turn, the invention provides a surface havingtarget molecules such as nucleic acids attached thereto by means of sucha reagent, as well as a material (e.g., microscope slide) that providessuch a surface.

[0030] Generally, the reagent molecules will first be attached to thesurface by activation of the photogroups, thereafter the targetmolecule, (e.g., a nucleic acid) is contacted with the bound reagentunder conditions suitable to permit it to come into binding proximitywith the bound polymer. The target molecule is thermochemically coupledto the bound reagent by reaction between the reactive groups of thebound reagent and appropriate functional groups on the target molecule.

[0031] The invention further provides a method of attaching a targetmolecule, such as a nucleic acid, to a surface, by employing a reagentas described herein. As used herein, with regard to describing thepresent invention, the word “oligonucleotide” (or “oligo”) shall referto a synthetic nucleic acid (as opposed to enzymaticaly prepared), andone that is typically shorter (e.g., on the order of 100-mer or less)than cDNA or PCR products formed enzymatically. The term “nucleic acid”,in turn, will refer to all such products collectively.

[0032] The invention further provides a surface having nucleic acidsattached thereto by means of such a reagent, as well as a material(e.g., a slide or microwell plate) that provides such a surface. In yetanother aspect, the invention provides a composition comprising areagent of this invention in combination with a target molecule thatcontains one or more functional groups reactive with thethermochemically reactive group of the reagent.

DETAILED DESCRIPTION

[0033] A preferred reagent composition of the present inventioncomprises a hydrophilic polymer bearing one or more pendent epoxidegroups adapted to form a covalent bond with corresponding reactivegroups of a target molecule, and also bearing one or more pendentphotoreactive groups adapted to be used for attaching the reagent to asurface, either before, during and/or after reaction between the reagentand the target molecules. Optionally, a composition can include othercomponents, in addition to the reagent polymer component, such aspolymers having pendent ionic groups, and the like.

[0034] In another embodiment of the invention, it is possible toimmobilize the reagent composition, and in turn the target molecules,without the use of the photoreactive group. For instance, the surface ofthe material to be coated can be provided with thermochemically reactivegroups, which can be used to immobilize hydrophilic polymers havingepoxide groups as described above. For example, a surface may be treatedwith an ammonia plasma to introduce a limited number of reactive amineson the surface of the material. If this surface is then treated with ahydrophilic polymer having epoxide groups, then the polymer can beimmobilized through reaction of the epoxide groups with amines on thesurface. Preferably, the concentration of epoxide groups on the polymeris in sufficient excess, relative to the concentration of amines on thesurface to insure that a sufficient number of reactive groups remainfollowing the immobilization to allow coupling with the nucleic acidsequence.

[0035] A polymeric backbone can be either synthetic or naturallyoccurring, and is preferably a synthetic copolymer of the epoxidemonomer and diluent or other monomers resulting from addition orcondensation polymerization. Naturally occurring polymers, such aspolysaccharides or polypeptides can be used as well. Preferred diluentmonomers are biologically inert, in that they do not provide abiological function that is inconsistent with, or detrimental to, theiruse in the manner described.

[0036] Suitable diluent monomers for use in preparing a reagent of thisinvention include acrylics such as hydroxyethyl acrylate, hydroxyethylmethacrylate, glyceryl acrylate, glyceryl methacrylate; acrylamidederivatives, such as acrylamide, methacrylamide, and acryloylmorpholine. Other synthetic polymers can be synthesized to includependent epoxide groups in the manner described herein, including vinylssuch as polyvinyl pyrrolidone and polyvinyl alcohol; nylons such aspolycaprolactam, polylauryl lactam, polyhexamethylene adipamide andpolyhexamethylene dodecanediamide; polyurethanes and polyethylene oxide,as well as combinations and copolymers thereof.

[0037] A reagent of the present invention preferably includes ahydrophilic polymer bearing a desired average number of photogroups andepoxide groups per average unit length or molecular weight, thecombination dependent upon the reagent selected. The epoxide monomer canalso be selected to provide any desired effective spacer distancebetween the polymeric backbone and the epoxide groups. In this manner,the reagent can be bonded to a surface or to an adjacent reagentmolecule, to provide the other groups with sufficient freedom ofmovement to demonstrate optimal activity. The diluent comonomers arepreferably hydrophilic (e.g., water soluble), with acrylamide andvinylpyrrolidone being particularly preferred.

[0038] Epoxide-containing polymers of the present invention can beprepared from monomers, such as glycidyl acrylate, glycidylmethacrylate, allylglycidyl ether, and glycidyl vinyl ether. Usefulmonomers are available from a variety of sources, for instance glycidylacrylate (Pfaltz & Bauer Chemicals, cat. # G03480), glycidylmethacrylate (Aldrich cat # 15,123-8), allylglycidyl ether (Aldrich, cat# A3,260-8), glycidyl vinyl ether (Aldrich, cat. # 45,865-1), andglycidyl vinylbenzyl ether (Aldrich cat. # 45,867-8).

[0039] Epoxide monomers can also be made, such as by reaction of2-isocyanatoethylmethacrylate with glycidol. Epoxide polymers can alsobe prepared by reacting hydroxyl polymers (e.g.,polyhydroxypropylacrylamide) with epichlorohydrin. Other epoxidemonomers and polymers can be made by those skilled in the art havingspacers of various lengths and with various polarities. Other examplesof suitable monomers are described in U.S. Pat. No. 5,763,629.

[0040] Epoxide-containing polymers can also be synthesized by reactinghydroxyl- or amine-containing polymers with diepoxides. Currently, anepoxide activated-Sepharose is available (Sigma) that is made byreacting Sepharose gel beads with 1,4-butanedioldiglycidyl ether. This,or other diepoxides, (e.g., ethylene glycol diglycidyl ether,diepoxyoctane or diepoxydecane) can be used for derivatizing eitheramine or hydroxyl polymers to make polyepoxides. For example,polyhydroxypropylacrylamide or a copolymer containing a photomonomer canbe reacted with an excess of 1,4-butanedioldiglycidyl ether to make apolyepoxide that can then be immobilized onto a surface.

[0041] Useful epoxide monomers include those of the general formula:

[0042] Where R₁ is either CH₃ or H and X is a noninterfering radical,preferably selected from the group:

[0043] where m=2-6 and n=1-10;

[0044] where n=1-10;

—(CH₂)_(m)-O—(CH₂)—

[0045] where m=0 or 1, and;

[0046] where m=1-20 and n=1-10.

[0047] Without intending to be bound by theory, it would appear thatepoxide groups can be used to immobilize underivatized DNA in a covalentfashion, and presumably due to a mechanism of reacting with amine groupson the purine and pyrimidine rings, such as cytosine and adenine, orwith terminal hydroxyl groups.

[0048] Reagents of the invention optionally carry one or more pendentlatent reactive (preferably photoreactive) groups covalently bonded tothe polymer backbone. Photoreactive groups are defined herein, andpreferred groups are sufficiently stable to be stored under conditionsin which they retain such properties. See, e.g., U.S. Pat. No.5,002,582, the disclosure of which is incorporated herein by reference.Latent reactive groups can be chosen that are responsive to variousportions of the electromagnetic spectrum, with those responsive toultraviolet and visible portions of the spectrum (referred to herein as“photoreactive”) being particularly preferred.

[0049] Photoreactive groups respond to specific applied external stimulito undergo active specie generation with resultant covalent bonding toan adjacent chemical structure, e.g., as provided by the same or adifferent molecule. Photoreactive groups are those groups of atoms in amolecule that retain their covalent bonds unchanged under conditions ofstorage but that, upon activation by an external energy source, formcovalent bonds with other molecules.

[0050] The photoreactive groups generate active species such as freeradicals and particularly nitrenes, carbenes, and excited states ofketones upon absorption of electromagnetic energy. Photoreactive groupsmay be chosen to be responsive to various portions of theelectromagnetic spectrum, and photoreactive groups that are responsiveto e.g., ultraviolet and visible portions of the spectrum are preferredand may be referred to herein occasionally as “photochemical group” or“photogroup”.

[0051] Photoreactive aryl ketones are preferred, such as acetophenone,benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles(i.e., heterocyclic analogs of anthrone such as those having N, O, or Sin the 10-position), or their substituted (e.g., ring substituted)derivatives. The functional groups of such ketones are preferred sincethey are readily capable of undergoing theactivation/inactivation/reactivation cycle described herein.Benzophenone is a particularly preferred photoreactive moiety, since itis capable of photochemical excitation with the initial formation of anexcited singlet state that undergoes intersystem crossing to the tripletstate. The excited triplet state can insert into carbon-hydrogen bondsby abstraction of a hydrogen atom (from a support surface, for example),thus creating a radical pair. Subsequent collapse of the radical pairleads to formation of a new carbon-carbon bond. If a reactive bond(e.g., carbon-hydrogen) is not available for bonding, the ultravioletlight-induced excitation of the benzophenone group is reversible and themolecule returns to ground state energy level upon removal of the energysource. Photoactivatible aryl ketones such as benzophenone andacetophenone are of particular importance inasmuch as these groups aresubject to multiple reactivation in water and hence provide increasedcoating efficiency. Hence, photoreactive aryl ketones are particularlypreferred.

[0052] The azides constitute a preferred class of photoreactive groupsand include arylazides (C₆R₅N₃) such as phenyl azide and particularly4-fluoro-3-nitrophenyl azide, acyl azides (—CO—N₃) such as benzoyl azideand p-methylbenzoyl azide, azido formates (—O—CO—N₃) such as ethylazidoformate, phenyl azidoformate, sulfonyl azides (—SO₂—N₃) such asbenzenesulfonyl azide, and phosphoryl azides (RO)₂PON₃ such as diphenylphosphoryl azide and diethyl phosphoryl azide. Diazo compoundsconstitute another class of photoreactive groups and includediazoalkanes (—CHN₂) such as diazomethane and diphenyldiazomethane,diazoketones (—CO—CHN₂) such as diazoacetophenone and1-trifluoromethyl-1-diazo-2-pentanone, diazoacetates (—O—CO—CHN₂) suchas t-butyl diazoacetate and phenyl diazoacetate, andbeta-keto-alpha-diazoacetates (—CO—CN₂—CO—O—) such as t-butyl alphadiazoacetoacetate. Other photoreactive groups include the diazirines(—CHN₂) such as 3-trifluoromethyl-3-phenyldiazirine, and ketenes(—CH═C═O) such as ketene and diphenylketene. Photoactivatible arylketones such as benzophenone and acetophenone are of particularimportance inasmuch as these groups are subject to multiple reactivationin water and hence provide increased coating efficiency.

[0053] Upon activation of the photoreactive groups, the reagentmolecules are covalently bound to each other and/or to the materialsurface by covalent bonds through residues of the photoreactive groups.Exemplary photoreactive groups, and their residues upon activation, areshown as follows. Photoreactive Group Residue Functionality aryl azidesamine R-NH-R′ acyl azides amide R-CO-NH-R′ azidoformates carbamateR-O-CO-NH-R′ sulfonyl azides sulfonamide R-SO₂-NH-R′ phosphoryl azidesphosphoramide (RO)₂PO-NH-R′ diazoalkanes new C-C bond diazoketones newC-C bond and ketone diazoacetates new C-C bond and esterbeta-keto-alpha- new C-C bond and diazoacetates beta-ketoester aliphaticazo new C-C bond diazirines new C-C bond ketenes new C-C bondphotoactivated ketones new C-C bond and alcohol

[0054] Copolymers can be prepared using epoxide-containing monomers asdescribed above, using techniques known to those skilled in the art.Preferably, the epoxide monomers and comonomers undergo free radicalpolymerization of vinyl groups using azo initiators such as2,2′-azobisisobutyronitrile (AIBN) or peroxides such as benzoylperoxide. The comonomers selected for the polymerization are chosenbased on the nature of the final polymer product. For example, aphotoreactive polymer containing epoxide groups can be prepared by theuse of a monomer mixture that includes one or more monomers containing aphotoreactive group and one or more second monomers containing anepoxide group.

[0055] An epoxide-functionalized polymeric reagent of this invention canbe prepared by appropriate derivatization of a preformed polymer or,more preferably, by polymerization of a set of comonomers to give thedesired substitution pattern. The latter approach is preferred becauseof the ease of changing the ratio of the various comonomers and by theability to control the level of incorporation into the polymer.

[0056] The composition of the final polymer can be controlled by moleratio of the monomers charged to the polymerization reaction. Typicallythe epoxide monomers are used at relatively low mole percentages of thetotal monomer content of the polymerization reaction, with the remainderof the composition consisting of a relatively low mole percent ofphotomonomers, and the remainder of monomers which are neitherphotoreactive nor thermochemically reactive toward the nucleic acidsequence. Examples of such monomers include, but are not limited to,acrylamide, acrylic acid and N-vinylpyrrolidone. Based on the relativereactivities of the monomers used, the distribution of the monomersalong the backbone is largely random.

[0057] In a preferred embodiment, for instance, a reagent composition isformed having between about 5 mole % and about 25 mole % epoxide monomer(more preferably between about 10 mole % and about 20 mole %); betweenabout 0.1 mole % and about 5 mole % photomonomer (more preferablybetween about 0.5 mole % and about 2 mole %), and the remainder (to 100mole %) other monomers.

[0058] The present invention provides a method and reagent compositionfor covalent attachment of target molecules onto the surface of asubstrate, such as slides formed of organosilane-pretreated glass,organosilane-pretreated silicon, silicon hydride, or plastic. In oneembodiment, the method and composition are used to immobilize nucleicacid probes onto plastic materials such as microwell plates, e.g., foruse in hybridization assays. In a preferred embodiment the method andcomposition are adapted for use with substantially flat or moldedsurfaces, such as those provided by organosilane-pretreated glass,organosilane-pretreated silicon, silicon hydride, or plastic (e.g.,polymethylmethacrylate, polystyrene, polycarbonate, polyethylene, orpolypropylene). The reagent composition can then be used to covalentlyattach a target molecule such as a biomolecule (e.g., a nucleic acid)which in turn can be used for specific binding reactions (e.g., tohybridize a nucleic acid to its complementary strand).

[0059] Support surfaces can be prepared from a variety of materials,including but not limited to plastic materials selected from the groupconsisting of crystalline thermoplastics (e.g., high and low densitypolyethylenes, polypropylenes, acetal resins, nylons and thermoplasticpolyesters) and amorphous thermoplastics (e.g., polycarbonates andpoly(methyl methacrylates). Suitable plastic or glass materials providea desired combination of such properties as rigidity, surfaceuniformity, resistance to long term deformation, and resistance tothermal degradation.

[0060] The present invention provides a method for immobilizingbiomolecules such as biopolymers, and particularly those selected fromnucleic acids, proteins, polysaccharides, the method comprising thesteps of:

[0061] a) providing a solid support having a surface,

[0062] b) providing a reagent comprising one or more epoxide groups, andoptionally also comprising one or more photogroups,

[0063] c) coating the reagent on the support surface (e.g., by dipping,spraying, roll-coating or knife-coating), and covalently attaching thepolymeric reagent to the support surface, e.g., by activation of thephotogroups,

[0064] d) providing a biopolymer (e.g., nucleic acid, protein,polysaccharide) having one or more corresponding thermochemical reactivegroups (e.g., amine, hydroxyl, sulfhydryl),

[0065] e) attaching the biopolymer to the support by reacting itscorresponding reactive group with the bound epoxide group,

[0066] f) optionally blocking the remaining unreacted epoxide groups(e.g., by the use of an amine reagent), and

[0067] g) using the resultant coated support surface for its intendedpurpose, e.g., immobilizing biomolecules, such as nucleic acids for usein hybridization (e.g., on slides for arrays, or in microplate wells),

[0068] In a preferred embodiment, the present invention provides amethod of attaching a target molecule to the surface of a substrate, themethod comprising:

[0069] a) providing a reagent composition comprising a polymericbackbone having one or more pendent epoxide groups adapted to formcovalent bonds with corresponding functional groups on the targetmolecule,

[0070] b) coating and immobilizing the reagent composition on thesubstrate surface,

[0071] c) providing a solution comprising a target molecule having oneor more functional groups thermochemically reactive with correspondingepoxide groups provided by the reagent composition,

[0072] d) applying an amount (e.g., in the form of discrete small samplevolume spots) of the solution on the surface of the substrate surface,and

[0073] e) incubating the combination under conditions suitable to permitthe epoxide groups provided by the reagent composition to form covalentbonds with corresponding functional groups provided by the targetmolecule in order to attach the target molecule to the surface.

[0074] Preferably, for use in preparing and using coated slides, thereagent is adapted to be coated and immobilized on a surface in a mannerthat permits:

[0075] i) a small sample volume of a solution containing the targetmolecule to be applied in the form of a discrete spot on thereagent-coated surface,

[0076] ii) target molecule present in the sample volume to becomecovalently attached to the bound reagent by reaction between itsfunctional groups and the epoxide groups, and

[0077] iii) substantially all unattached target molecule to be washedfrom the spot without undue detectable amounts of target molecule in thearea surrounding the spot.

[0078] In a preferred embodiment, the target molecules are preferablyapplied to the epoxide polymer-coated surface in a solution atrelatively low ionic strength and slightly alkaline pH (e.g., in 150 mMphosphate buffer, pH 8.5). Optimal coupling can be achieved byincubating the surface at high humidity (e.g., 75% relative humidity),which can be achieved by placing the surface within an enclosed storagebox containing saturated NaCl at room temperature) for several hours, ormost preferably, overnight. The excess uncoupled epoxide groups are thenblocked with a solution containing 50 mM ethanolamine and 0.1% sodiumdodecyl sulfate (SDS) in 0.1M Tris buffer, pH 9 for 30 minutes at 50° C.The surface is then rinsed with deionized water and washed with4×standard saline citrate (“SSC”)(0.6 M NaCl+0.06 M sodium citrate)containing 0.1% SDS at 50° C. for about 15 to about 60 minutes. Thesurface is then rinsed with deionized water and spun dry in acentrifuge.

[0079] When used for preparing microarrays, e.g., to attach captureprobes (e.g., oligonucleotides or cDNA) to the microarray surface, suchcapture probes are generally delivered to the surface in a volume ofless than about 1 nanoliter per spot, using printing pins adapted toform the spots into arrays having center to center spacing of about 200μm to about 500 μm. Unlike the coupling of DNA from solution and ontothe surface of coated microplate wells, nucleic acids printed in arraysof extremely small spot sizes tend to dry quickly, thereby altering theparameters affecting the manner in which the nucleic acids contact andcouple with the support. In addition to the design and handling of theprinting pins, other factors can also affect the spot size and/or theultimate hybridization signals, including: salt concentrations, type ofsalts and wetting agents in the printing buffer; hydrophobic/hydrophilicproperties of the surfaces; the size and/or concentration of the nucleicacids; and the drying environments.

[0080] In a preferred embodiment, the reagent composition can be used toprepare activated slides having the reagent composition photochemicallyimmobilized thereon. The slides can be stably stored and used at a laterdate to prepare microarrays by immobilizing underivatized oramine-derivatized DNA. The coupling of the capture DNA to the surfacetakes place at pH 8-9 in a humid environment following printing the DNAsolution in the form of small spots.

[0081] Activated slides of the present invention are particularly wellsuited to replace conventional (e.g., amino-silylated) glass slides inthe preparation of microarrays using manufacturing and processingprotocols, reagents and equipment such as micro-spotting robots (e.g.,as available from Cartesian), and a micro-spotting device (e.g., asavailable from TeleChem International). Suitable spotting equipment andprotocols are commercially available, such as the “ArrayIt”™ ChipMaker 3spotting device. This product is said to represent an advanced versionof earlier micro-spotting technology, employing 48 printing pins todeliver as many as 62,000 samples per solid substrate (e.g., 1 inch by 3inch standard slide).

[0082] The use of such an instrument, in combination with conventional(e.g., poly-L-lysine coated) slides, is well known in the art. See, forinstance, U.S. Pat. No. 5,087,522 (Brown et al.) “Methods forFabricating Microarrays of Biological Samples”, and the references citedtherein, the disclosures of each of which are incorporated herein byreference.

[0083] For instance, the method and system of the present invention canbe used to provide a substrate, such as a coated glass slide, with asurface having one or more microarrays. Each microarray preferablyprovides at least about 10/cm² (and preferably at least about 100/cm²distinct target molecules (e.g., polynucleotide or polypeptidebiopolymers). Each distinct target molecule 1) is disposed at aseparate, defined position in the array, 2) has a length of at least 10subunits, 3) is present in a defined amount between about 50 attomolesand about 10 nanomoles, and 4) is deposited in selected volume in thevolume range of about 0.01 nanoliters to about 100 nanoliters. Theseregions (e.g., discrete spots) within the array can be generallycircular in shape, with a typical diameter of between about 75 micronsand about 1000 microns (and preferably between about 100 and about 200microns). The regions are also preferably separated from other regionsin the array by about the same distance (e.g., center to center spacingof about 100 microns to about 1000 microns). A plurality ofanalyte-specific regions can be provided, such that each region includesa single, and preferably different, analyte specific reagent (“targetmolecule”).

[0084] Those skilled in the art, given the present description, will beable to identify and select suitable reagents depending on the type oftarget molecule of interest. Target molecules include, but are notlimited to, plasmid DNA, cosmid DNA, expressed sequence tags (ESTs),bacteriophage DNA, genomic DNA (includes, but not limited to yeast,viral, bacterial, mammalian, insect), RNA, complementary DNA (cDNA),peptide nucleic acid (PNA), and oligonucleotides.

[0085] The invention will be further described with reference to thefollowing non-limiting Examples. It will be apparent to those skilled inthe art that many changes can be made in the embodiments describedwithout departing from the scope of the present invention. Thus thescope of the present invention should not be limited to the embodimentsdescribed in this application, but only by embodiments described by thelanguage of the claims and the equivalents of those embodiments. Unlessotherwise indicated, all percentages are by weight. Structures of thevarious “Compounds” identified throughout these Examples can be found inTable 1 included below.

EXAMPLE 1 Preparation of 4-Benzoylbenzoyl Chloride (BBA-Cl) (Compound I)

[0086] 4-Benzoylbenzoic acid (BBA), 1.0 kg (4.42 moles), was added to adry 5 liter Morton flask equipped with reflux condenser and overheadstirrer, followed by the addition of 645 ml (8.84 moles) of thionylchloride and 725 ml of toluene. Dimethylformamide, 3.5 ml, was thenadded and the mixture was heated at reflux for 4 hours. After cooling,the solvents were removed under reduced pressure and the residualthionyl chloride was removed by three evaporations using 3×500 ml oftoluene. The product was recrystallized from 1:4 toluene:hexane to give988 g (91% yield) after drying in a vacuum oven. Product melting pointwas 92-94° C. Nuclear magnetic resonance (NMR) analysis at 80 MHz (¹HNMR (CDCl₃)) was consistent with the desired product: aromatic protons7.20-8.25 (m, 9H). All chemical shift values are in ppm downfield from atetramethylsilane internal standard. The final compound was stored foruse in the preparation of a monomer used in the synthesis ofphotoactivatable polymers as described, for instance, in Example 3.

EXAMPLE 2 Preparation of N-(3-Aminopropyl)methacrylamide Hydrochloride(APMA) (Compound II)

[0087] A solution of 1,3-diaminopropane, 1910 g (25.77 moles), in 1000ml of CH₂Cl₂ was added to a 12 liter Morton flask and cooled on an icebath. A solution of t-butyl phenyl carbonate, 1000 g (5.15 moles), in250 ml of CH₂Cl₂ was then added dropwise at a rate which kept thereaction temperature below 15° C. Following the addition, the mixturewas warmed to room temperature and stirred 2 hours. The reaction mixturewas diluted with 900 ml of CH₂Cl₂ and 500 g of ice, followed by the slowaddition of 2500 ml of 2.2 N NaOH. After testing to insure the solutionwas basic, the product was transferred to a separatory funnel and theorganic layer was removed and set aside as extract #1. The aqueous layerwas then extracted three times with 1250 ml of CH₂Cl₂, keeping eachextraction as a separate fraction. The four organic extracts were thenwashed successively with a single 1250 ml portion of 0.6 N NaOHbeginning with fraction #1 and proceeding through fraction #4. This washprocedure was repeated a second time with a fresh 1250 ml portion of 0.6N NaOH. The organic extracts were then combined and dried over Na₂SO₄.Filtration and evaporation of solvent to a constant weight gave 825 g ofN-mono-t-BOC-1,3-diaminopropane which was used without furtherpurification.

[0088] A solution of methacrylic anhydride, 806 g (5.23 moles), in 1020ml of CHCl₃ was placed in a 12 liter Morton flask equipped with overheadstirrer and cooled on an ice bath. Phenothiazine, 60 mg, was added as aninhibitor, followed by the dropwise addition ofN-mono-t-BOC-1,3-diaminopropane, 825 g (4.73 moles), in 825 ml of CHCl₃.The rate of addition was controlled to keep the reaction temperaturebelow 10° C. at all times. After the addition was complete, the ice bathwas removed and the mixture was left to stir overnight. The product wasdiluted with 2400 ml of water and transferred to a separatory funnel.After thorough mixing, the aqueous layer was removed and the organiclayer was washed with 2400 ml of 2 N NaOH, insuring that the aqueouslayer was basic. The organic layer was then dried over Na₂SO₄ andfiltered to remove drying agent. A portion of the CHCl₃ solvent wasremoved under reduced pressure until the combined weight of the productand solvent was approximately 3000 g. The desired product was thenprecipitated by slow addition of 11.0 liters of hexane to the stirredCHCl₃ solution, followed by overnight storage at 4° C. The product wasisolated by filtration and the solid was rinsed twice with a solventcombination of 900 ml of hexane and 150 ml of CHCl₃. Thorough drying ofthe solid gave 900 g ofN-[N′-(t-butyloxycarbonyl)-3-aminopropyl]-methacrylamide, m.p. 85.8° C.by DSC. Analysis on an NMR spectrometer was consistent with the desiredproduct: ¹H NMR (CDCl₃) amide NH's 6.30-6.80, 4.55-5.10 (m, 2H), vinylprotons 5.65, 5.20 (m, 2H), methylenes adjacent to N 2.90-3.45 (m, 4H),methyl 1.95 (m, 3H), remaining methylene 1.50-1.90 (m, 2H), and t-butyl1.40 (s, 9H).

[0089] A 3-neck, 2 liter round bottom flask was equipped with anoverhead stirrer and gas sparge tube. Methanol, 700 ml, was added to theflask and cooled on an ice bath. While stirring, HCl gas was bubbledinto the solvent at a rate of approximately 5 liters/minute for a totalof 40 minutes. The molarity of the final HCl/MeOH solution wasdetermined to be 8.5 M by titration with 1 N NaOH using phenolphthaleinas an indicator. TheN-[N′-(t-butyloxycarbonyl)-3-aminopropyl]methacrylamide, 900 g (3.71moles), was added to a 5 liter Morton flask equipped with an overheadstirrer and gas outlet adapter, followed by the addition of 1150 ml ofmethanol solvent. Some solids remained in the flask with this solventvolume. Phenothiazine, 30 mg, was added as an inhibitor, followed by theaddition of 655 ml (5.57 moles) of the 8.5 M HCl/MeOH solution. Thesolids slowly dissolved with the evolution of gas but the reaction wasnot exothermic. The mixture was stirred overnight at room temperature toinsure complete reaction. Any solids were then removed by filtration andan additional 30 mg of phenothiazine were added. The solvent was thenstripped under reduced pressure and the resulting solid residue wasazeotroped with 3×1000 ml of isopropanol with evaporation under reducedpressure. Finally, the product was dissolved in 2000 ml of refluxingisopropanol and 4000 ml of ethyl acetate were added slowly withstirring. The mixture was allowed to cool slowly and was stored at 4° C.overnight. Compound II was isolated by filtration and was dried toconstant weight, giving a yield of 630 g with a melting point of 124.7°C. by DSC. Analysis on an NMR spectrometer was consistent with thedesired product: ¹H NMR (D₂O) vinyl protons 5.60, 5.30 (m, 2H),methylene adjacent to amide N 3.30 (t, 2H), methylene adjacent to amineN 2.95 (t, 2H), methyl 1.90 (m, 3H), and remaining methylene 1.65-2.10(m, 2H). The final compound was stored for use in the preparation of amonomer used in the synthesis of photoactivatable polymers as described,for instance, in Example 3.

EXAMPLE 3 Preparation of N-[3-(4-Benzoylbenzamido)propyl]methacrylamide(BBA-APMA) (Compound III)

[0090] Compound II 120 g (0.672 moles), prepared according to thegeneral method described in Example 2, was added to a dry 2 liter,three-neck round bottom flask equipped with an overhead stirrer.Phenothiazine, 23-25 mg, was added as an inhibitor, followed by 800 mlof chloroform. The suspension was cooled below 10° C. on an ice bath and172.5 g (0.705 moles) of Compound I, prepared according to the generalmethod described in Example 1, were added as a solid. Triethylamine, 207ml (1.485 moles), in 50 ml of chloroform was then added dropwise over a1-1.5 hour time period. The ice bath was removed and stirring at ambienttemperature was continued for 2.5 hours. The product was then washedwith 600 ml of 0.3 N HCl and 2×300 ml of 0.07 N HCl. After drying oversodium sulfate, the chloroform was removed under reduced pressure andthe product was recrystallized twice from 4:1 toluene: chloroform using23-25 mg of phenothiazine in each recrystallization to preventpolymerization. Typical yields of Compound III were 90% with a meltingpoint of 147-151° C. Analysis on an NMR spectrometer was consistent withthe desired product: ¹H NMR (CDCl₃) aromatic protons 7.20-7.95 (m, 9H),amide NH 6.55 (broad t, 1H), vinyl protons 5.65, 5.25 (m, 2H),methylenes adjacent to amide N's 3.20-3.60 (m, 4H), methyl 1.95 (s, 3H),and remaining methylene 1.50-2.00 (m, 2H). The final compound was storedfor use in the synthesis of photoactivatable polymers as described, forinstance, in Examples 5 and 6.

EXAMPLE 4 Synthesis of a Spaced Epoxide Monomer (Compound IV)

[0091] To three ml of chloroform was added isocyanatoethylmethacrylate(1.0 ml, 7.04 mmole), glycidol (0.50 ml, 7.51 mmole) and triethylamine(50 μl, 0.27 mmole). The reaction was stirred at room temperatureovernight. The product was purified on a silica gel column and thestructure confirmed by NMR. The yield was 293 mg (18% yield).

EXAMPLE 5 Synthesis of a Copolymer of Acrylamide, BBA-APMA and SpacedEpoxide Monomer (Compound V)

[0092] Acrylamide (1.12 gm, 15.7 mmoles), BBA-APMA (30 mg, 0.085 mmole)and spaced epoxide monomer (Compound IV) (273 μl, 1.28 mmole) weredissolved in 15.6 ml of tetrahydrofuran (THF). To this solution wasadded 34 mg of 2,2′-azobisisobutyronitrile (AIBN) and 16 μl ofN,N,N′,N′-tetramethylethylenediamine (TEMED). The solution was spargedwith helium for four minutes, then argon added to head space, thentightly capped and placed in a 55° C. oven overnight. The reactionmixture containing precipitated polymer was centrifuged and thesupernatant decanted. The residue was resuspended in 20 ml of fresh THF,centrifuged and decanted. This was repeated, followed by filtering andfurther washing of the polymer with two ten ml aliquots of THF. Thepolymer was the dried under vacuum to a constant weight. The yield was1.477 gm.

EXAMPLE 6 Preparation of Copolymer of Acrylamide, BBA-APMA, and GlycidylMethacrylate (Photo PA-Polyepoxide) (Compound VI)

[0093] Acrylamide (7.1 gm, 99.35 mmoles), BBA-APMA (0.414 gm, 1.18mmole) and 2,2′-azobis(2-methylbutyronitrile) (“VAZO 67”, 0.262 gm, 1.4mmole) were dissolved in 108 ml of THF. To this solution was added 2.4ml of glycidylmethacrylate (17.7 mmoles). The solution was sparged withhelium for four minutes, then with argon for four minutes, then cappedtightly and heated at 61° C. overnight while mixing. The polymer wascollected by filtration, then suspended in methanol and mixed, afterwhich it was again collected by filtration and washed with chloroform,then dried in a vacuum oven at 30° C.

EXAMPLE 7 Preparation of Microscope Slides Coated with PolyEpoxide

[0094] Soda lime glass microscope slides (Erie Scientific, Portsmouth,N.H.) were silane treated by dipping in a mixture ofp-tolyldimethylchlorosilane (T-Silane) and N-decyldimethylchlorosilane(D-Silane, United Chemical Technologies, Bristol, Pa.), 1% each inacetone, for 1 minute. After air drying, the slides were cured in anoven at 120° C. for one hour. The slides were then washed with acetonefollowed by DI water dipping. The slides were further dried in an ovenfor 5-10 minutes.

[0095] Compound VI was sprayed onto the silane treated slides, whichwere then illuminated using a Dymax lamp (25 mjoule/cm² as measured at335 nm with a 10 nm band pass filter on an International Lightradiometer) while wet, washed with water, and dried.

EXAMPLE 8 Preparation and Use of Microarrays with PhotoPA-PolyepoxideCoated Slides

[0096] PCR products derived from actin, glucose phosphate dehydrogenase(GPDH) or β-galactosidase genes were printed onto slides at 0.1 mg/ml in0.15 M phosphate buffer using an X, Y, Z motion controller to positionChipMaker 2 microarray spotting pins (Telechem International). Theslides were either coated with Photo-PA-polyepoxide as in Example 7 orpolylysine slides prepared by published methods (See U.S. Pat. No.5,087,522 (Brown et al.) “Methods for Fabricating Microarrays ofBiological Samples”, and the references cited therein). The printedepoxide slides were incubated overnight at room temperature and 75%relative humidity. The printed polylysine slides were UV crosslinked.After printing, the epoxide slides were blocked with 50 mM ethanolaminein 0.1M tris buffer, pH 9.0 and washed. The polylysine slides wereprocessed by the published procedure. Both types of slides werehybridized with Cy5-labeled total RNA either spiked with β-gal mRNA at1:250,000 or not spiked. The slides were then scanned with a laserscanner to measure intensities of Cy5 fluorescence. Example 9 Noβ-Galactosidase Spike β-Galactosidase Spike Actin GPDH β-Gal Actin GPDHβ-Gal Epoxide 33913 ± 5984 ± 211 ± 47351 ± 29007 ± 9571 ± Coating  4370 443  27  2170  8825 2531 Poly-L- 29946 ± 4751 ± 282 ± 34836 ± 11545 ±4524 ± Lysine  1805  686  32  2222  1882  825

Preparation and Use of Microarrays with Photo-polyepoxide Coated Slides

[0097] Oligonucleotides, either aminated or nonaminated, were printedonto slides at 8 μM in 0.15 M phosphate buffer using an X, Y, Z motioncontroller to position ChipMaker 2 microarray spotting pins (TelechemInternational). The slides were either coated with photo-PA-polyepoxideas in Example 7, with photo-PA-PolyNOS by the same procedure or withpolylysine by published methods (See references in Example 8). Theprinted epoxide and NOS slides were incubated overnight at roomtermperature and 75% relative humidity. The printed polylysine slideswere processed by the published procedure. The slides were scanned tomeasure the Cy3 fluorescence of immobilized capture oligos, thenhybridized at 41° C. overnight with Cy5-labeled oligonucleotide (1pmole/slide) that was complementary to the capture oligos. The slideswere scanned with a laser scanner to measure the fluorescenceintensities of the hybridized oligos. Because the amount of captureoligo immobilized with the amine-silane slides was so low, they were nothybridized. Capture Oligo Hybridization Immobilized Signal Coated Amine-Non-amine- Amine- Non-amine- Slides oligo oligo oligo oligo NOS 30,302 ±5037 ± 25,226 ± 6116 6090 ± 503 3866 294 Epoxide 36,793 ± 30,821 ±26,526 ± 10887 28,467 ± 11695 5145 3585 Amino- 800 ± 492 ± ND ND silane85 92

[0098] TABLE 1 Compounds

COMPOUND I

COMPOUND II

COMPOUND III

COMPOUND IV

COMPOUND V

[0099] (where x=0.1 to 5 mole %, y=2 to 30 mole % and z=65 to 97.9 mole%)

Compound VI

[0100] (where x=0.1 to 5 mole %, y=2 to 30 mole % and z=65 to 97.9 mole%)

What is claimed is:
 1. A reagent composition for attaching a targetmolecule to the surface of a substrate, the reagent compositioncomprising a polymeric backbone adapted to be covalently attached to thesurface and comprising one or more pendent epoxide groups adapted toform covalent bonds with corresponding functional groups on the targetmolecule.
 2. A reagent composition according to claim 1 wherein thereagent comprises a polymer formed by the polymerization of one or moremonomers selected from the group glycidyl acrylate, glycidylmethacrylate, allylglycidyl ether, and glycidyl vinyl ether.
 3. Areagent composition according to claim 1 wherein the reagent comprises apolymer formed by the polymerization of one or more monomers of theformula:

where R₁ is either CH₃ or H and X is a noninterfering radical,preferably selected from the group:

where m=2-6 and n=1-10;

where n=1-10; —(CH₂)_(m)-O—(CH₂)— where m=0 or 1, and;

where m=1-20 and n=1-10.
 4. A reagent composition according to claim 1wherein the reagent comprises a polymer synthesized by reactinghydroxyl- or amine-containing polymers with diepoxides.
 5. A reagentcomposition according to claim 1 wherein the target molecule comprises anucleic acid and the surface comprises the surface of a support formedof organosilane-pretreated glass, organosilane-pretreated silicon,silicon hydride, or plastic.
 6. A reagent composition according to claim5 wherein the nucleic acid comprises an underivatized nucleic acid.
 7. Areagent composition according to claim 6 wherein the underivatizednucleic acid comprises an oligonucleotide.
 8. A reagent compositionaccording to claim 1 wherein the composition further comprises one ormore latent reactive groups comprising photoreactive groups forcovalently attaching the reagent composition to the surface uponapplication of energy from a suitable source.
 9. A reagent compositionaccording to claim 8 wherein the target molecule is a nucleic acid andthe photoreactive groups are selected from the group consisting ofphotoreactive aryl ketones.
 10. A reagent composition according to claim1 wherein the polymeric backbone is selected from the group consistingof acrylics, vinyls, nylons, polyurethanes and polyethers, and thebackbone further comprises one or more pendent photoreactive groupsselected from the group consisting of aryl ketones.
 11. A method ofattaching a target molecule to the surface of a substrate, the methodcomprising: a) providing a reagent composition according to claim 1, b)coating and immobilizing the reagent composition on the substratesurface, c) providing a solution comprising a target molecule having oneor more functional groups thermochemically reactive with correspondingepoxide groups provided by the reagent composition, d) applying anamount of the solution on the substrate surface, and e) allowing theepoxide groups provided by the reagent composition to form covalentbonds with corresponding functional groups provided by the targetmolecule in order to attach the target molecule to the surface.
 12. Amethod according to claim 11 wherein the reagent comprises a polymerformed by the polymerization of one or more monomers selected from thegroup glycidyl acrylate, glycidyl methacrylate, allylglycidyl ether, andglycidyl vinyl ether.
 13. A method according to claim 11 wherein thereagent comprises a polymer formed by the polymerization of one or moremonomers of the formula:

where R₁ is either CH3 or H and X is a noninterfering radical,preferably selected from the group:

where m=2-6 and n=1-10;

where n=1-10; —(CH₂)_(m)-O—(CH₂)— where m=0 or 1, and;

where m=1-20 and n=1-10.
 14. A method according to claim 11 wherein thereagent comprises a polymer synthesized by reacting hydroxyl- oramine-containing polymers with diepoxides.
 15. A method according toclaim 11 wherein the target molecule comprises a nucleic acid and thesurface comprises the surface of a support formed oforganosilane-pretreated glass, organosilane-pretreated silicon, siliconhydride, or plastic.
 16. A method according to claim 15 wherein thenucleic acid comprises an underivatized nucleic acid.
 17. A methodaccording to claim 16 wherein the underivatized nucleic acid comprisesan oligonucleotide.
 18. A method according to claim 11 wherein thecomposition further comprises one or more latent reactive groupscomprising photoreactive groups for attaching the reagent composition tothe surface upon application of energy from a suitable source.
 19. Amethod according to claim 11 wherein the method is used to prepare oneor more microarrays of nucleic acids upon the surface of a slide formedof organos ilane-pretreated glass, organosilane-pretreated silicon,silicon hydride, or plastic, each microarray providing at least about10/cm² distinct nucleic acids having a length of at least 10nucleotides, the nucleic acids each being spotted in discrete regionsand defined amounts of between about 50 attomoles and about 10nanomoles.
 20. A method according to claim 19 wherein the regions aregenerally circular in shape, having a diameter of between about 75microns and about 1000 microns and separated from other regions in thearray by center to center spacing of about 100 microns to about 1000microns.
 21. An activated slide comprising a flat support surface coatedwith the bound residue of a reagent composition according to claim 1.22. An activated slide according to claim 21 wherein the slide isadapted for fabricating a microarray wherein the target moleculecomprises a nucleic acid and the surface comprises the surface of aslide formed of organosilane-pretreated glass, organosilane-pretreatedsilicon, silicon hydride, or plastic.
 23. An activated slide accordingto claim 21 wherein the slide provides at least about 10/cm² distinctnucleic acids having a length of at least 10 nucleotides, the nucleicacids each being spotted in discrete regions and defined amounts ofbetween about 50 attomoles and about 10 nanomoles.
 24. An activatedslide according to claim 23 wherein the regions are generally circularin shape, having a diameter of between about 75 microns and about 1000microns and separated from other regions in the array by center tocenter spacing of about 100 microns to about 1000 microns.
 25. Amicroarray prepared by a method comprising: a) providing a reagentcomposition according to claim 1, b) coating and immobilizing thereagent composition on the substrate surface, c) providing a solutioncomprising a target molecule comprising a nucleic acid having one ormore functional groups thermochemically reactive with epoxide groupsprovided by the reagent composition, d) applying one or more discretesmall sample volume spots of the solution on the surface of thesubstrate surface, and e) allowing the epoxide groups provided by thereagent composition to form covalent bonds with corresponding functionalgroups provided by the target molecule in order to attach the targetmolecule to the surface.
 26. A microarray according to claim 25 whereinthe microarray provides at least about 10/cm² distinct nucleic acidshaving a length of at least 10 nucleotides, the nucleic acids each beingspotted in discrete regions and defined amounts of between about 50attomoles and about 10 nanomoles.
 27. A microarray according to claim 26wherein the regions are generally circular in shape, having a diameterof between about 75 microns and about 1000 microns and separated fromother regions in the array by center to center spacing of about 100microns to about 1000 microns.
 28. A microarray according to claim 25wherein the reagent comprises a polymer formed by the polymerization ofone or more monomers of the formula:

where R₁ is either CH₃ or H and X is a noninterfering radical,preferably selected from the group:

where m=2-6 and n=1-10;

where n=1-10; —(CH₂)_(m)-O—(CH₂)— where m=0 or 1, and;

where m=1-20 and n=1-10, or by the reaction of hydroxyl- oramine-containing polymers with diepoxides, the polymeric backbone isselected from the group consisting of acrylics, vinyls, nylons,polyurethanes and polyethers, the backbone further comprises one or morependent photoreactive groups selected from the group consisting of arylketones, the nucleic acid comprises an underivatized oligonucleotides,the surface comprises the surface of a slide formed oforganosilane-pretreated glass, organosilane-pretreated silicon, siliconhydride, or plastic, the microarray provides at least about 10/cm²distinct nucleic acids having a length of at least 10 nucleotides, thenucleic acids each being spotted in discrete regions and defined amountsof between about 50 attomoles and about 10 nanomoles, and the regionsare generally circular in shape, having a diameter of between about 75microns and about 1000 microns and separated from other regions in thearray by center to center spacing of about 100 microns to about 1000microns.